Reducing the toxicity of agrobacterium endotoxin

Abstract

The present invention relates to the fields of genetically modified Agrobacterium strains, vaccine adjuvants, and generally molecular biology and immunology. Provided herein are modified Agrobacterium strains that produce lipopolysaccharide (LPS) having reduced toxicity or detoxified lipopolysaccharide, and methods of obtaining such strains for plant-based production of biologies. Also provided herein are uses of reduced or detoxified LPS as adjuvants suitable for clinical use.

We claim: 
     
       1. A genetically modified strain of  Agrobacterium , wherein the genetically modified strain comprises a functional deletion of one or more polypeptides required for synthesis of Very Long Chain Fatty Acid (VLCFA) and is deficient in Lipopolysaccharide (LPS) toxicity relative to its parent strain;
 wherein the functional deletion is achieved by removal of at least a portion of the gene encoding AcpXL-dependent lipid A acyltransferase (lpxXL) and at least a portion of the gene encoding Acyl carrier protein (acpXL), and 
 wherein the genetically modified strain comprises an exogenous nucleic acid encoding Lipid A 4′-phosphatase (LpxF), and 
 wherein the genetically modified bacterium retains the ability to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into a plant cell genome. 
 
     
     
       2. The modified strain of  claim 1 , wherein the modified strain produces tetra-acylated lipid A lacking VLCFA. 
     
     
       3. The modified strain of  claim 1 , wherein the modified strain produces penta-acylated lipid A with VLCFA replaced by a 16:0 acyl chain or 18:1 acyl chain. 
     
     
       4. The modified strain of  claim 1 , wherein the modified strain produces a mixture of tetra- and penta-acylated lipid A. 
     
     
       5. The modified strain of  claim 1 , wherein the parent strain is  A. tumefaciens  GV3101 or  A. tumefaciens  LBA4404. 
     
     
       6. The modified strain of  claim 1 , wherein the parent strain is  A. tumefaciens  strain C58. 
     
     
       7. A method or reducing Lipopolysaccharide (LPS) toxicity in an  Agrobacterium  bacterium, the method comprising: deleting or disrupting at least a portion of a gene encoding AcpXL-dependent lipid A acyltransferase (IpxXL) and at least a portion of a gene encoding Acyl carrier protein (acpXL) in the bacterium, wherein the deletion or disruption comprises a functional deletion;
 introducing an exogenous nucleic acid encoding Lipid A 4′-phosphatase (LpxF) into the bacterium; whereby a genetically modified  Agrobacterium  is obtained; 
 wherein the genetically modified  Agrobacterium  exhibits lower LPS toxicity than an unmodified  Agrobacterium , and 
 wherein the genetically modified  Agrobacterium  retains the ability to mediate T-DNA transfer into plant cells, and to mediate T-DNA insertion into a plant cell genome. 
 
     
     
       8. The method of  claim 7 , wherein the  Agrobacterium  bacterium is  Agrobacterium tumefaciens  strain GV3101 or LBA4404. 
     
     
       9. The method of  claim 7 , wherein the  Agrobacterium  bacterium is  Agrobacterium tumefaciens  strain C58. 
     
     
       10. A method for generating a LPS variant molecule having reduced endotoxicity and improved immunogenicity, the method comprising the steps of:
 (a) providing the modified  Agrobacterium  strain of  claim 1 ; 
 (b) allowing the modified  Agrobacterium  strain to grow under conditions to produce a LPS variant molecule having reduced endotoxicity and improved immunogenicity compared to an LPS molecule relative to its parent strain. 
 
     
     
       11. A lipopolysaccharide (LPS) isolated from a genetically modified  Agrobacterium  of  claim 1 , wherein the LPS exhibits reduced toxicity relative to LPS produced by a wild-type  Agrobacterium  or an  Agrobacterium  that is not so genetically modified. 
     
     
       12. An adjuvant comprising an immunostimulatory quantity of the reduced toxicity LPS of  claim 11  and a pharmaceutically acceptable carrier.